bioind. and biomark.-2011 cap. livro

7/28/2019 Bioind. and Biomark.-2011 Cap. Livro

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Bioindicators and Biomarkers in theAssessment of Soil Toxicity

Carmem Silvia Fontanetti, Larissa Rosa Nogarol, Raphael Basto de Souza,Danielli Giuliano Perez and Guilherme Thiago Maziviero

Department of Biology, Institute of Biosciences, So Paulo State University (UNESP)Rio Claro, SP,

Brazil

1. Introduct ion

Several potentially harmful chemical compounds, derived from activities of urban centres,rural properties and industries are constantly released into the terrestrial environment. Inthis context, the scientific community has shown increasing interest in the detection,knowledge and control of environmental agents responsible for damages to human healthand sustainability of ecosystems (Magalhes & Ferro-Filho, 2008).Monitoring the types and quantities of toxic substances that are entering into the terrestrialenvironment is an exhaustive and problematic task due, mainly, to the complexity and costresulting from the identification of the chemical substances involved. Despite the numerous

analytical methods available, collecting sufficient samples in a timely fashion continues to bea great obstacle in the evaluation of environmental damages (Silva et al., 2003).Furthermore, the determination of isolated substances by traditional chemical analyses has alimited environmental application, since it does not detect the effects on the organismsneither inform about the possible interactions between the substances (additive, antagonisticor synergistic), as well as their bioavailability (Magalhes & Ferro-Filho, 2008). In thissense, researchers have pointed the necessity to apply biological methodologies in order toobtain an ecosystemic approach.Biological factors may indicate better the environmental balance through the biotic indexes,derived from the observation of bioindicator species. According to Hodkinson and Jackson(2005), it is called bioindicator a species or group of species that reflects biotic and abiotic

levels of contamination of an environment, presenting alterations that enables thegeneration of information about the quality of the environment, for example, accumulatingsubstances in concentrations higher than those considered normal or essential for its bodymetabolism or presenting alterations in the number of organisms. Such organisms, due totheir characteristics of little ecological tolerance to some chemical substances, can presentsome alteration, whether it is physiological, morphological of behavioural, when exposed tocertain pollutants (Magalhes & Ferro-Filho, 2008).Due to their close contact with soil, some taxonomic groups of invertebrates belonging to themeso- and macro-fauna such as, Isopoda, Collembola, Oligochaeta and Diplopoda, havebeen proposed as bioindicator organisms (Hopkin, 2002). In addition, higher plants such asAllium cepa (onion),Arabidopsis thaliana (mustard), Hordeum vulgare (barley), Tradescantia sp.,


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Soil Contamination144

Vicia faba (broad bean) and Zea mays (corn) are also commonly used in the assessment of soiltoxicity, particularly for cytotoxicity, genotoxicity and mutagenicity assays (White &Claxton, 2004).In a biological system, the sequential order of alterations promoted by the presence of

pollutants occurs in crescent levels of biological organization, extending from the molecular orbiochemical level to the physiological or individual level, until the population and ecosystemlevel (Stegeman et al., 1992). When a significative alteration is evident, the ecosystem must bealready severely damaged. Therefore, techniques that show responses at lower levels ofbiological organization are considered more preventive (Nascimento et al., 2008).Morphological alterations can be used as biomarkers in toxicity investigations of specificchemical compounds and in the monitoring of the acute and chronic effects of organismsexposed to impacted environments. In this context, the morphological analysis of targetorgans, carried out by ultra-morphology, histology and ultrastructure, has become widelyused in studies with invertebrates, aiming to identify different damages caused by harmfulsubstances to the organisms (Fontanetti et al., 2010).

Another tool that has shown to be increasingly efficient in the assessment of soil toxicants onthe organisms is the use of molecular biomarkers. Recent studies show great interest in theuse of enzymatic biomarkers as a form to monitor the environment, since the increase orinhibition of the activity of certain enzymes can explain a possible response to theenvironment stress.Due to the importance to ensure the genetic integrity of the organisms, biomarkers ofgenotoxicity are gaining attention in the evaluation of the toxic potential of soil samples(Misik et al., 2011). The tests used in the genotoxic assessment of an agent (genotoxicity andmutagenicity tests), include the Ames test, chromosome aberrations test, micronucleus test,comet assay, SMART test (Somatic Mutation and Recombination Test), microarray andmicroscreen, using techniques of cellular, molecular and genetic biology both in vitro and in

vivo, in situ and ex situ.Given the above, the aim of the present chapter was to compile and discuss informationpresent in the literature about the use of animal and plant bioindicators in the analysis ofsoil toxicity, as well as characterize the different biomarkers used in these organisms thatenable the assessment of the soil toxicant effects in different levels of the biological scale, i.e.,morphological, biochemical and genotoxic.

2. Complex substances, organic compounds and metals: potential soi lcontaminants

Population growth combined with the increasing industrialization is responsible forgenerating tons of waste per day, which, many times, are accumulated in the environmentwithout any previous treatment. Soil becomes a cheaper and practice alternative for the finaldisposal of these residues, but not without consequences. Soil contamination is a broadproblem, since the contaminants can be leached into groundwater, rivers and lakes. Themajor xenobiotics responsible for the contamination of this compartment as well as theirimplications for invertebrates and plants will be discussed.

2.1 Vinasse

Among the substances released into the soil with toxic potential it can be cited the vinasse, aproduct of the alcohol production, composed by water (97%) and a solid fraction (organicmatter and mineral elements). According to Sahai et al. (1985), due to the fast growth of the


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Bioindicators and Biomarkers in the Assessment of Soil Toxicity 145

distilleries, there was, consequently, an increase in the amount of this residue, which wastraditionally discharged in open areas or next to water courses, causing air, water and soilpollution. For Junior et al. (2008), the reuse of previously treated vinasse as fertilizer or soilconditioner becomes an alternative of great interest. However, Lyra et al. (2003) affirm that

there are only few studies that evaluate its pollutant potential on soil and groundwater.Thus, studies about the effects of vinasse application in the soil have been developed byresearchers of different countries. Recently, Brazilian researchers have studied thegenotoxicity and mutagenicity of vinasse applied in natura in the soil or associated withother compounds. Souza et al. (2009) usedA. cepa to evaluate the mitotic and chromosomeabnormalities resulted from exposure to landfarming soil treated with vinasse, used as apossible bioremediator. According to the authors, the vinasse was responsible forpotentiating the clastogenicity of the landfarming by decreasing the pH and, thus, makingavailable the metals that were strongly adsorbed in the organic matter of the soil.Other studies on the vinasse toxicity were conducted by Christofoletti and Fontanetti (2010)and Pedro-Escher and Fontanetti (2010) also usingA. cepa as test organism. In the first study,the preliminary results show that the vinasse did not present cytotoxicity nor mutagenicity,but it presented genotoxic potential when applied in natura or associated with sewagesludge samples; the second study showed that different concentrations of vinasse diluted inwater (12.5%, 25% and 50%) presented genotoxic potential and only the raw vinassepresented mutagenic potential, thus suggesting that the vinasse can cause damages in thegenetic material of certain organisms.

2.2 Sewage sludge

Another residue with pollutant potencial and problems in final disposal is the sewage

sludge generated in the STSs (Sewage Treatment Stations). Its great production, mainly in

the large urban centres, has led researchers to intensify the studies about the use of thesewaste with agricultural purposes. Therefore, recycling, via agriculture use, presents itself as

a global trend (Lopes et al., 2005).

Nevertheless, sewage sludge can present, in its composition, undesirable chemicals (metalsand organic chemical compounds) and biological elements (pathogens) that, in contact with

man and/or fauna and flora, may cause contamination and diseases. Thus, any decision on

the most appropriate final destination depends on the evaluation and minimization of thecontamination risks of the environment and man (Rocha & Shirota, 1999).

Studies involving millipeds exposed to sewage sludge have shown that its components can

affect the integrity of organs such as the midgut of these animals (Godoy & Fontanetti, 2010;Nogarol & Fontanetti, 2010, 2011; Perez & Fontanetti, 2011a). Mazzeo et al. (2010), usingA.cepa, investigated the genotoxic and mutagenic potential of domestic sewage sludge at

different concentrations.

2.3 Polycycl ic aromatic hydrocarbons (PHAs)

Some of the main pollutants that cause concern in relation to soil contamination are thePAHs (Bispo et al., 1999), which are compounds formed by two or more benzene rings,exclusively constituted by atoms of carbon and hydrogen (Netto et al., 2000). There aremany origin sources but it can be highlighted industrial processes, such as petroleumrefining, combustion of organic matter and burning of coal (Page et al., 1999). According tothe IUPAC (International Union of Pure and Applied Chemistry) there are, currently, over


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100 known PAHs, however only 16 have environmental and toxicological importance(Environment Protection Agency [EPA], 1986). These compounds are able to react, directlyor after undergoing metabolic transformations, with the DNA, becoming potentialcarcinogens and efficient mutagens.

Soil receives considerable amounts of PAHs that, because of the complexity of theirchemical structure, low solubility in water and strong sorption tendency into the soil,become recalcitrant and remain for long periods in the environment, enhancing theprobability of exposure of humans and animals to these compounds (Jacques et al., 2007).When present in the environment they can be transferred to invertebrates by ingestion ofsoil and plant material contaminated or by cuticle (Achazi & Van Gestel, 2003).In order to reduce the negative impact of these compounds in the soil, petroleum refineriesuse a bioremediation system called landfarming. The technique has been used for thetreatment of soils contaminated with hydrocarbons for 100 years and, the petroleumindustry for at least 25 years (Riser-Roberts, 1998). Currently, other types of industry startedto employ this technique, such as textile and food industries and treatment of effluents.Souza et al. (2009) carried out bioassays withA. cepa in order to assess landfarming soilsamples before and after biodegradation of hydrocarbons. Before biodegradation, thelandfarming had 13.5 g/Kg of Total Petroleum Hydrocarbons (TPH) and caused strongclastogenic and mutagenic effects. After 108 days of biodegradation, the concentration ofTPH decreased 27% with significant reduce of mitotic and chromosome abnormalities,micronuclei and nuclear buds.Using the diplopod R. padbergi exposed to different concentrations of industrial soilcontaminated with PAHs, Souza et al. (2011) and Souza and Fontanetti (2011) analyzed theperivisceral fat body and midtgut of the animals and verified that there were severalalterations in these two tissues.

2.4 DioxinsSeven dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs) and 12polychlorinated biphenyls (PCBs) are called dioxins (Word Health Organizatrion [WHO],2010; United States Environmental Protection Agency [USEPA], 2000, 2003), being releasedinto the environment as a byproduct of chemical processes and through the combustion ofindustrial and municipal wastes (Stephens et al., 1995). According to Schlatter (1994), sincethe accident in Seveso, Italy, they became the symbol of threat caused by toxic chemicals. Asa result of widespread fear, dioxins are a matter of real concern in relation to environmentalcontamination.Among the isomers of PCDDs, the most toxic is 2,3,7,8 - tetrachloro-dibenzo-para-dioxin

(2,3,7,8-TCDD) (Eisler, 1986). There is little information in literature on the effects of PCDDson terrestrial invertebrates. Reinecke and Nash (1984) reported that two species ofearthworms (Allolobophophora caliginosa and Lumbricus rubellus) showed no adverse effectswhen exposed for 85 days in soil with 5 ppm of 2,3,7,8-TCDD, but both species died at 10ppm. Studies involving plants and PCBs have been made, since these organisms are lesssensitive to PCBs and thus may be a possible route of biomagnification in various foodchains (Sinkkonen et al., 1995).

2.5 Agrochemicals

The use of fertilizers and pesticides has become a common practice due to populationgrowth, food crisis and consequent need for the increase in the agriculture production.


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Within the existing agriculture model, agrochemicals are classified as one of the mainchemical pollutants that are disseminated throughout the planet (Grisolia, 2005). However,there is still little information about the effects of these chemical compounds oninvertebrates that occupy levels of high sensitivity in trophic chain (Mantecca et al., 2006).

Among the studies carried out with terrestrial invertebrates it was analyzed the possiblealterations in the biomass (Niemeyer et al., 2006a), reproduction (Helling et al., 2000),behaviour (Niemeyer et al., 2006b), survival (Diao et al., 2007) and tissular and cellularlesions (Nasiruddin & Mordue, 1993) resulted from exposure to certain agrochemicals.Associated with the use of bioindicators and biomarkers it is also used the direct analysis ofthe presence of residues in soil samples by specific equipments such as thespectrophotometer or chromatograph.Pesticides have been widely tested by bioassays with plants and positive results are usuallyobtained (Leme & Marin-Morales, 2009). A clear example is theA. cepa test successfully usedin the evaluation of the mutagenic and genotoxic potential of herbicides such as trifluralin(Fernandes et al., 2007; 2009).

2.6 MetalsHeavy metals or trace metals are terms applied for a great amount of trace elements that areindustrially and biologically important. From the point of view of human health, agricultureand ecotoxicology, the most worrying heavy metals are As, Cd, Hg, Pb, Ti and U. Studiesinvolving heavy metals in ecosystems have shown that many areas near urban centres,mines and road systems have high concentrations of these elements (Alloway, 1994). Metalsare highly persistent in the soil with persistence of up to thousands of years (McGrath, 1987)and can express their pollutant potential directly on the soil organisms by availability toplants and transference to the food chain, both by plants and by the contamination ofsuperficial waters or groundwater (Chang et al., 1987).

The main anthropogenic sources of metals are fertilizers, pesticides, contaminated irrigationwater, combustion of coal and oil, vehicular emissions, incineration of urban and industrialwastes and, mainly, mining and smelting (Tavares & Carvalho, 1992).Due to their habits in the superficial layers of the soil, invertebrates of the saprophagousfauna, such as isopods, diplopods and springtails are regularly exposed to metals (Hopkin,2002). Heikens et al. (2001) carried out a literature review to clarify the concentration ofmetals in terrestrial invertebrates and they concluded that the concentration in most of thegroups happened in the order Pb>Cd>Cu. Afterwards, Khler (2002) conducted a study todetermine the location of these metals in the bodies of soil arthropods. The genotoxicpotential of metals has also been studied by several authors using plants as test systems(Knasmller et al., 1998; Rank & Nielsen, 1998).

3. Invertebrates of the edaphic fauna and higher plants as soil b ioindicators

One important question in ecotoxicological studies refers to the choice of the bioindicatorspecies. It will depend on its ecological and toxicological importance, facility to bemaintained in laboratory, reproductive rate and sensitivity (it must be affected by severalchemical agents but less affected by abiotic factors) (Rmbke & Garcia, 2000). Many authorsagree that the main features needed to be a good bioindicator are sensitivity, goodrepresentativeness and functional importance in the ecosystem, as well as easy collection,identification and analysis (Greensdale, 2007). In this context, some taxonomic groups of soilinvertebrates and higher plants have been proposed as bioindicator organisms.


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3.1 Terrestrial invertebrates

Terrestrial arthropods of the saprophagous fauna such as, Isopoda, Collembola andDiplopoda are among the most appropriate organisms to evaluate the effects of theaccumulation of toxic substances present in the soil, due to their direct contact with

contaminants present in it (Grff et al., 1997; Hopkin et al., 1989). Annelids, in special theOligochaeta, are also frequently used in toxicity tests. These invertebrates get in contact witha great variety of pollutants present in this compartment by their movement and ingestionof contaminated soil or leaf litter (Spadotto et al.2004).Oligochaeta are considered one of most important representatives of the edaphic macro-fauna (Kale, 1988). Several factors make earthworms excellent bioindicators of the toxicity ofchemical substances in the soil, such as the knowledge already accumulated on theirhabitats and important trophic position of these invertebrates, which are situated in thelowest levels of the terrestrial food webs, serving as food for several animals and route oftransference and biomagnification of contaminants along these webs (Andra, 2010).Due to their great importance in the soil, their wide distribution and all the reasons

previously cited, earthworms, mainly the species Eisenia fetida (figure 1) and E. andrei werechosen for several toxicity tests for registration of agrochemicals in the regulatory agenciesof several countries, including Brazil (Andra, 2010). Other species such as Lumbricusterrestris and L. rubellus have been widely used in studies of bioaccumulation of metals(Amaral & Rodrigues, 2005; Veltman et al., 2007).

Fig. 1. Earthworm Eisenia fetida. (Photo: Raphael Basto de Souza and Larissa Rosa Nogarol)

Collembola are among the most important members of the soil meso-fauna involved in thedecomposition process and are vulnerable to the effects of its contamination (Bengtsson &Rundgren, 1984). Greensdale (2007) lists some favourable points in choosing Collembola as

bioindicators, such as presence in all ecosystems, abundance and ease of collection insufficient number to allow statistical analyses. Moreover, they have short life cycle, makingthat they respond quickly to environmental changes and, as they are in direct contact withthe soil, they are more sensitive to some type of stress applied in the ecosystem.Several studies point out this organism as bioindicator, applying different methodologiesand evaluation parameter. Tests of reproduction associated to survival rates (Pedersen et al.,2009; Sverdrup et al., 2010;) and evaluation of abundance and/or diversity of species inareas that suffer some type of degradation (Sousa et al., 2004) are the most usedmethodologies.Another taxonomic group used in toxicological analyses is Isopoda, one of the largest ordersof crustaceans with approximately 10,000 thousand described species, mostly marine


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(Schultz, 1982). Terrestrial isopods have already been used in toxicity tests of soil and themain parameters of evaluation were abundance of individuals (Faulkner & Lochmiller,2000), reproduction rates (Niemeyer et al., 2009) and survival (Stanek et al., 2006).Metals are the main toxic agents evaluated using Isopoda, since these invertebrates

bioaccumulate these elements. In this sense, researchers have carried out studies onbioaccumulation (Blanusa et al., 2002; Hopkin et al., 1993), the cytotoxic effect of metals(Khler et al., 1996a; Odendaal & Reinecke, 2003) and the detoxification mechanisms (Hopkin,1990; Khler & Triebskorn, 1998) and the terrestrial isopodPorcellio scaberis the most studied.The importance of diplopods in the recycling of nutrients, aeration and fertilization of soil isfrequently mentioned in the literature (Dangerfield & Telford, 1989). Due to the habits of thediplopods, colonizers of various soil layers, these animals can be greatly influenced by thedeposition of metals, organic compounds and complex substances in the soil.Most studies in the literature using diplopods as bioindicators of the soil are related tometals. However, the effect of organic pollutants and complex mixtures on theseinvertebrates is relatively little known (Souza & Fontanetti, 2011). In this context, the first

study carried out with diplopods, as possible bioindicators, was conducted by Hopkin et al.(1985), involving the assimilation of metals by the species Glomeris marginata. In this study, itwas verified a higher uptake of copper, zinc and cadmium by the animals collected in soilscontaminated when compared to those animals collected in non-contaminatedenvironments. The authors comment that ultrastructural studies of different organs wouldbe necessary to understand the metals path, particularly in the gut of these invertebrates.Later, Triebskorn et al. (1991) exposed several invertebrates such as mites, insects anddiplopods to different toxic substances and used the ultrastructural analysis in order todemonstrate the applicability of using such animals in biomonitoring. In the study carriedout by Khler et al. (1992), it was analyzed the impact of lead on the efficiency ofassimilation in diplpods, submitted to different environmental conditions. The researchers

used different species of diplopods and found that only Glomeris conspersa increased theingestion of food containing lead when compared to a non-contaminated diet.Recently, the toxicity assessment of complex substances was performed with the Brazilianspecies Rhinocricus padbergi (figure 2) exposed to different concentrations of sewage sludge(Godoy & Fontanetti, 2010; Nogarol & Fontanetti, 2010, 2011; Perez & Fontanetti, 2011a) andlandfarming (Souza & Fontanetti, 2011; Souza et al., 2011). The histological andhistochemical analysis, as well as the ultrastructural analysis, showed that such substancesare toxic to the diplopod studied, since different tissular and cellular alterations wereobserved in the midgut and perivisceral fat body of these invertebrates.

Fig. 2. Diplopod Rhinocricus padbergi. (Photo: Larissa Rosa Nogarol and Raphael Basto deSouza)


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3.2 Higher p lants

Plants, despite their structure and metabolic differences, can offer important informationabout the cytotoxic, genotoxic and mutagenic potential of substances, even when exposed inshort term and offer some advantages such as low cost cultivation and easy maintenance,

comparatively, to mammals (Rodrigues et al., 1997). In studies with complex mixtures,plants have also shown satisfactory results, indicating that plants are sensitive enough todetect the adverse effects of environmental samples (Majer et al., 2005).Plants can be directly exposed to the contaminant, without any dilution or filtration of thesample (Steinkellner et al., 1999). Moreover, Grant (1994) cites other advantages ofemploying higher plants: (1) higher plants are eukaryotes, thus, their structure and cellularorganization are similar to that of humans and it is possible to establish comparisons withanimals; (2) the techniques employed for the study are relatively simple and can beperformed with agility; (3) cultivation of the organisms has low cost and easy maintenance;(4) the assays can be carried out under a wide range of environmental conditions, pH andtemperature; (5) higher plants can regenerate easily; (6) assays with higher plants can be

used to assess the genotoxic potential of simple substances or even complex mixtures; (7) itcan be used for in situ monitoring; (8) can be used for monitoring for several years and arehighly reliable; (9) studies have shown correlations with cytogenetic assays in mammals;(10) can be used together with microbial assays to detect mutagenic metabolites (pro-mutagens); (11) genotoxicity studies with plants are presenting high sensitivity in tests withcarcinogenic agents.On the other hand, according to Majer et al. (2005), one of the limitations of using plants asbioindicators is the lack of sensitivity for certain classes of pro-mutagens such as thenitrosamines, heterocyclic amines and some classes of PAHs. In contrast, Ventura (2009)showed that the A. cepa system is susceptible to nitro aminobenzene, while Mazzeo (2009)observed the same effect for benzene, toluene, ethylbenzene and xilene (BTEX).Among the higher plants, onion (A. cepa) is the most used plant to determine the cytotoxic,genotoxic and mutagenic effects of many substances present in the soil. Its cellular kineticscharacteristic favours a rapid growth of the roots, due to the great number of cells indivision. Therefore, the record of the mitotic activity and abnormalities in the cell cycle ofthe meristematic cells of its roots can be easily visualized (Grant, 1994). Leme and Marin-Morales (2009) affirm that theA. cepa test is a fast and sensitive technique to detect genoxoticand mutagenic substances dispersed in the environment.The evaluation of genetic alteration can be also performed using different species of thegenus Tradescantia (figure 3) through the detection of mutations induced by agents presentin the air, soil and water by the analysis of micronuclei in the mother cell of the pollen grain

(Trad-MCN). The species Tradescantia are specially indicated for direct application in regionsand countries in development due to the advantages such as easy handling and relativelylow maintenance cost (Shima et al., 1997).Vicia faba is a popular material that has been widely used not only in cytological studies, butalso in physiological experiments (Kanaya et al., 1994). This organism was initially used inradiobiological tests in investigation of mechanisms of formation of chromosomalaberrations by ionizing radiation (Read, 1959). Later, Kihlman (1975) developed andstandardized the V. faba meristematic cell bioassay for analysis of chromosomal aberrations,and since then has been widely used for genotoxicity studies for evaluation of sisterchromatid exchange (Kihlman & Kronborg, 1976; Kihlman & Andersson, 1984). Thistechnique is very similar to A. cepa test; the method does not require sterile conditions or


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any material or equipment of high cost. Further details of this test are described by Kihlman(1975).

Fig. 3. Tradescantia pallida. (Photo: Guilherme Thiago Maziviero)

4. Biomarkers

Molecular, biochemical and physiological compensatory mechanisms can become operativein organisms after exposure to environmental contaminants. This may result in theinhibition or facilitation of one or more physiological mechanisms or functional andstructural changes. In this sense, the use of biomarkers allows obtaining information aboutthe biological effects of pollutants and mechanisms of action of xenobiotics on the fauna.Several authors have proposed different definition for the term biomarkers. According toLam and Gray (2003), biomarkers can be defined as biochemical, cellular or molecularalterations or physiological changes in the cells, body fluids, tissues or organs of anorganism that are indicative of exposure or effect of a xenobiotic. Despite being older, the

definition proposed by Depledge (1993) and Depledge et al. (1993) has a morecomprehensive character and it is considered the most widely used nowadays: biomarkersare defined as adaptive biological responses to stressors, evidenced as biochemical, cellular,histological, physiological or behavioural alterations.In the scope of measuring the toxic effects in the organisms at a cellular or molecular level,biomarkers represent an initial response to environmental disturbances and contamination.Therefore, they are generally considered more sensitive than the tests that measure theseeffects at higher levels of biological hierarchy, such as individual or population (McCarthy& Shugart, 1990).Thus, during the last decades, several biomarkers have been used effectively, especially astests for specific toxicants, since biomarkers when combined with biomonitors can create asophisticated multiple target system to detect a variety of environmental hazards in a fastand economically feasible way, in a single test organism, helping in the establishment ofpriorities for action in the control of environmental pollution.

4.1 Morphological biomarkers

The detection of many classes of damage in several tissues and cellular types becomespossible by using morphological biomarkers. Such morphological alterations may providequalitative evidences of a functional adaptation to the external environment (Meyers &Hendricks, 1985). Moreover, the qualitative assessment of such changes before the death ofthe organism may provide early indications of toxicity (Nogarol & Fontanetti, 2010;Triebskorn et al., 1999).


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For such analysis, histology and ultrastructure are used. By these techniques it is possible todiagnose cellular and sub-cellular symptoms resulted from intoxication as well as locatesymptoms of cellular death and reveal reactions in response to chronic and sub-lethalexposure in cells and tissues (Fontanetti et al., 2010; Kammenga et al., 2000).

Studies show that one of the main contaminants of the soil, metals, are selectivelyconcentrated in only one or few organs, or in specific regions of the tissues in most of soilinvertebrates and typically these organs are part of the digestive tract (Dallinger, 1993). Forexample, in millipeds (Khler & Alberti, 1992), isopods (Dallinger & Prosi 1988) andspringtails (Pawert et al., 1996), the epithelium of the midgut is the main target of metals.Thus, the epithelium of the digestive tract represents the first barrier against the intoxicationof the whole organism (Walker, 1976).In diplopods, some studies with this approach were performed using the digestive tube andthe fat body (Hopkin et al., 1985; Khler & Triebskorn, 1998; Triebskorn et al., 1999).Morphological alterations observed in the midgut (figure 4) and in the perivisceral fat body(figure 5) of the diplopod R. padbergi were successfully used as sublethal biomarkers in the

evaluation of soils contaminated with complex substances such as sewage sludge (Godoy &Fontanetti, 2010; Nogarol & Fontanetti, 2010, 2011; Perez & Fontanetti, 2011a) andlandfarming (Souza & Fontanetti, 2011; Souza et al., 2011).In the studies performed with the diplopod R. padbergi, it was possible to observe tissularand cellular responses related to detoxification mechanisms such as increased cytoplasmicgranules (spherocrystals) and intense release of secretory vesicles into the intestinal lumenof these invertebrates (Nogarol & Fontanetti, 2010; Perez & Fontanetti, 2011a). Thesesecretory vesicles of the apocrine type seems to help in the detoxification of toxic substancesinitially absorbed by the organism and form a protector layer that would reduce the contactbetween the toxic agent and the intestinal epithelium.The formation of agglomerates of haemocytes through the cells of the fat body layer was

also observed and this response is directly related to a defence mechanism of the animal.According to van de Braak (2002), haemocytes can migrate to the injury site in the tissue bya chemotaxis process that results in inflammation. By this inflammatory reaction, these cellsact in the removal of toxins and possibly help in the re-absorption of the damagedepithelium in order to maintain the homeostasis of the organism. In a recent reviewconducted by Perez and Fontanetti (2011b) it becomes clear that this tissular response iscommon in different invertebrates exposed to environmental stress conditions. According tothe authors, the monitoring of the number of haemocytes can be used as a measure of stressin sentinel species due to environmental contamination.The mechanisms of defence and detoxification require high and continuous energyexpenditure, especially when the organism is exposed to a toxic agent for a long period. In

this sense, histological and ultrastructural studies showed some of the main responses ofthis invertebrate related to higher energetic needs. Nogarol and Fontanetti (2011) observedat an ultrastructural level a high increase in the number of tracheioles between the cells ofthe fat body layer that compose the midgut of diplopods sub-chronically exposed tosewage sludge. The authors suggest that a higher oxygenation of the tissue was necessary toenable the formation of molecules of adenosine triphosphate (ATP), used in thedetoxification mechanisms.Toxic agents may be able to cause cellular death by necrosis, evidenced mainly by theintense cytoplasmic vacuolization in the principal cells of the midgut epithelium ofdiplopods exposed to landfarming (Souza & Fontanetti, 2011) and sewage sludge (Nogarol& Fontanetti, 2011; Perez & Fontanetti, 2011a). In addition to the cytoplasm, other cellular


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compartments were affected by the exposure to toxic agents leading to cellular inviability. Inthese cases, damaged cells are expelled towards intestinal lumen.Samples of landfarming and sewage sludge presented genotoxic action, evidenced by theoccurrence of nucleus fragmentation in the principal epithelial cells, karyolysis in the

nucleus of the cells of the fat body layer (Souza & Fontanetti, 2011) and loss of integrity ofthe nuclear envelope of hepatic cells and cells of the fat body layer of the midgut (Nogarol& Fontanetti, 2011).

Fig. 4. Midgut of the diplopod R. padbergi stained with Hematoxylin-Eosin. Unexposedanimals (A; B); Animals exposed to sewage sludge (C-H). secretion vesicles (C); haemocytesaglomeration (D); epithelium renewal (E); increase of cytoplasmatic granules in fat bodylayer (F); cytoplasmatic vacuolization (G); volume reduction of the cells in fat body layerof midgut (H). e=epithelium; m= muscle layer; fb= fat body layer; h= haemocytes; v=vacuole; sv= secretion vesicle; * dilatation of intercellular space (Photos: Larissa Rosa

Nogarol; Raphael Basto de Souza and Tatiana da Silva Souza)

Fig. 5. Perivisceral fat body of the diplopod R. padbergi stained with Hematoxylin-Eosin (A;B) and submitted to TEM routine (C; D). Unexposed animal (A); Animal exposed to sewagesludge (B-D). Loss of cell limit and increase of spherocrystal (B); Cytoplasmaticvacuolization and loss of cell membrane integrity (C); Nucleus deformation (D). t=trophocyte; tr = tracheoles; o= oenocyte; m= mitochondria; n= nucleus; v= vacuole; arrows=spherocrystals; *= loss of cell membrane integrity. (Photos: Raphael Basto de Souza andLarissa Rosa Nogarol)


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In diplopods, the fat body, both parietal and perivisceral, is constituted of trophocytes andoenocytes (Fontanetti et al., 2004) and presents intense metabolic activity, such as storage oflipids, uric acid and proteins as well as storage, neutralization and excretion of substancesthat are not useful (Fontanetti et al., 2006; Hopkin & Head, 1992; Hubert, 1979). In this

sense, Souza et al. (2011) exposed diplopods of the species R. padbergi in bioassayscontaining industrial soil contaminated by PAHs and metals (landfarming) in order toanalyze histological and histochemical alterations in the perivisceral fat body. The authorsconcluded that the fat body can be used as a target organ and that the alterations observed,such as loss of integrity of the plasmatic membrane, cytoplasmic disorganization anddepletion of energetic reserves can be considered stress biomarkers in this animal. Similarresponses were observed in animals exposed to sewage sludge (Abe et al., 2010).

4.2 Genotoxic ity biomarkers

The increase in the genotoxic load in the terrestrial ecosystems by the release of chemicalproducts and physical agents can cause impact on the organisms, inducing increase in thefrequency of mutations; such effects can lead to a decrease in the size of the population and,eventually, extinction of species and consequently affect the stability of this ecosystem(Majer et al., 2005). In this sense, it became necessary to develop different tests to evaluatethe genotoxic potential of soil samples.Due to the highly conserved structure of the genetic material, it is possible to use a widevariety of species in genotoxicity tests; currently, the most widespread methods for theroutine tests are based on the use of indicator bacteria and also basidiomycetes fungi, plants,insects and cultured mammalian cells or even laboratory animals for mutagenicity tests.According to the literature, the Ames test is the most widely used in genotoxicityevaluations of soils and leachate (Claxton et al., 2010; Wlz et al., 2011). This test, also

known as Salmonella/microsome, consists, basically, in the employment of strains of theauxotrophic bacteria Salmonella typhimurium, i.e., deficient in the synthesis of the aminoacidhistidine; the strains of these cells are unable to grow in minimum medium, where themutagenic compounds are able to restore the synthesis capacity of this aminoacid, thus, themutagenic expression corresponds to the growth of the colony in a minimum culturemedium and it can be easily detected by counting the colonies (Umbuzeiro & Vargas, 2003).However, due to the low sensitivity of the Ames test for heavy metals, more studies shouldbe directed to the development of bioassays with higher organisms (Gatehouse et al., 1990,as cited in Lah et al., 2008).Meristematic cells of A. cepa and V. faba, for example, constitute an effective cytogeneticmaterial to analyze chromosome aberrations (figure 6) caused by soil pollution. The use of

meristematic cells makes possible the quantification of several morphological andcytogenetic parameters (endpoints), including the morphology and growth of roots anddetermination of several parameters of cytotoxicity, genotoxicity and mutagenicity. Theanalysis of the cytotoxicity can be done by determining the mitotic index and cell death. Theinduction of aberrant metaphases, anaphases and telophases, such as bridges, loss andchromosome stickiness, polyploidy, irregular nuclei and nuclear buds are parameters forthe genotoxicity analysis, while the micronuclei and chromosome breaks allow themutagenicity analysis (Fernandes et al., 2007; Leme & Marin-Morales, 2008; Souza et al.,2009).Chromosome aberration test concerns the discovery of the mechanisms of action of aparticular agent, since the division process is well known. Kovalchuk et al. (1998) state that


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the chromosome aberrations assay with A. cepa, can be used as a tool for quantifying andmonitoring genetic alterations in soils radioactively contaminated. Moreover, thechromosome aberration assay in onion was the first of nine plant systems accepted in theGenotoxic Program of the Environmental Protection Agency (USEPA) and is widely used

for monitoring residual water. Such sensitivity is attributed by Ma et al. (1995), to the largesize of the chromosomes and because they are mostly metacentric. On the other hand,bioassays based on chromosome aberrations, in certain cases, tend to be replaced by lesstime-consuming techniques, such as the micronucleus assay (MN), which can be performedwith mitotic cells in roots (of V. faba or A. cepa) or meiotic cells, in tetrads of Tradescantia(Misik et al., 2011).

Fig. 6. Alterations observed inA. cepa meristematic cells. (A) nuclear bud (arrow head) andmicronucleus (arrow); (B) micronucleus (arrow); (C) C-metaphase; (D) anaphase withchromossomal bridge (arrows); (E) telophase with cromossomal bridge (arrow) andchromossomal break (arrows head); (F) telophase with cromossomal delay (arrow); (G) celldeath. (Photos: Cintya Aparecida Christofoletti)

The micronucleus test in Tradescantia (Trad-MCN) (figure 7) is a sensitive mutagenicity test,of short exposure and simple evaluation, applicable in the species T. pallida and in the clonesBNL 4430 and KU 20 (Misik et al., 2011). Besides the Trad-MCN, it is possible to evaluatemutations in somatic cells of the staminal hair (Trad-SHM) in young inflorescences of the

hybrid clone BNL 4430 (Brookhaven National Laboratory). However, currently, the cloneKU 20 (Kyoto University) is more applicable to this technique due to the higher number ofinflorescences per cycle. The mutation results in the expression of the recessive allele, whichimplies in the phenotype of pink colouration. The high rate of pink cells, as well as the lossof reproductive capacity are indicative of mutagenicity (Ma et al., 1996).

Fig. 7. Micronucleus in polen cells of Tradescantia (arrow in B). (Photos: Janana Pedro-Escher)

4.3 Molecular biomarkers

The use of molecular biomarkers in the environment monitoring represents a significativetool for the evaluation of the contamination in different organisms. Despite morphologicalmarkers provide good qualitative evidence of damages caused by certain pollutants, it isknown that biochemical alterations resulted from the toxic action of a contaminant are earlyevidence of negative effects of the exposure, since they precede the onset of visible damages.


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Following, it is presented some biochemical biomarkers extensively used in studies ofenvironmental impact, including ecotoxicological analyses of the soil.

4.3.1 Enzymatic antioxidants

The use of enzymatic activity as biomarker is due to the fact that toxic compounds have highaffinity for electron pairs found in the aminoacids that form the enzymes (Cogo et al., 2009).One of the main monitored parameters in ecotoxicological analyses of the soil is theconcentration of metals. Exposure to metals can intensify the production of reactive oxygenspecies (ROS), which are normally produced in non-stressed cells and their excess can leadto the oxidative stress and cause harmful effects (Barreiros et al., 2006).When a cell undergoes oxidative damage, the injuries are minimized in the differentorganisms by enzymatic and non-enzymatic antioxidants (Freitas et al., 2008). Among theenzymatic antioxidants, some examples would be the superoxide dismutase, catalase,gluthathione reductase and gluthathione -S-transferase (Mishra et al., 2006).Superoxide dismutase catalyzes the formation of H2O2 from O2. This enzyme was the firstdiscovered among the enzymatic antioxidants and, generally is one of the first to act againstdamages caused by ROS (Nordberg & Arnr, 2001).Now, the catalase function is to facilitate the removal of H2O2, degrading it in H2O and O2.Thus, it reduces the risk of forming the radical hydroxyl from H2O2, since this oxygenreactive species is one of the most harmful to the biological systems (Betteridge, 2000;Diplock et al., 1998).The metabolism of gluthathione is one of the main antioxidant defence mechanisms in theliving systems (Valko et al., 2006) and specific metals can induce the synthesis of thiscompound in different species (Backor et al., 2007). In order to perform its function asoxidant agent, gluthathione must be in its reduced form, reaction catalyzed the enzyme

gluathathione reductase (Creissen et al., 1994).Another important defence system against the increase of free radicals involves the enzyme

gluthathione peroxidase, which acts in the removal of hydrogen peroxide and lipid

peroxides from the cell (Rover Junior et al., 2001). One of the forms of the gluthathione

peroxidase is the gluthatione-S-transferase, one of the most studied detoxicant enzymes indifferent organisms, since it has an essential role in the cellular response to the stress caused

by herbicides in plants. It is considered a detoxification enzyme because it metabolizes agreat variety of xenobiotic compounds, catalyzing their conjugation with the reduced

molecule of gluthathione and forming substances of low toxicity (Malmezat et al., 2000).

According to Almeida (2003), depending on the type of contaminant and exposure period ofthe organism to the contaminated environment, the activity of the antioxidant enzymes canbe stimulated or inhibited. Generally, the increase of the enzyme activity results from an

increase in the production of ROS, which leads to a exacerbated induction of enzymes; now,the decrease can be related to prolonged exposure of the organism to environments highlycontaminated, where the production of ROS and the consequent deleterious effects of suchproduction surpasses the defence efforts of the organism.Several other studies describe alterations in the enzymatic activities of the superoxide

dismutase, catalyse, gluthathione reductase and gluthathione peroxidise in differentorganisms exposed to stress conditions, especially metals, corroborating their use as

effective biochemical biomarkers in the evaluation of environmental impacts (Bocchetti etal., 2008; Cogo et al., 2009).


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4.3.2 Heat shock proteins

All organisms, from bacteria to mammals, respond to different environmental stressconditions by the synthesis of highly conserved proteins, known as heat shock proteins(HSPs) (Hamer et al., 2004). They are so called because they were first described in cells of

Drosophila melanogaster during exposure to high temperatures. At the time, it was verifiedthat the exposure of cells to heat produced a new pattern of thickening of chromosomes,which represented specific sites of transcription for the synthesis of proteins. The stressinduced the expression of certain genes, which led the cell to produce a certain class ofproteins, so-called heat shock proteins. Later, researchers observed that these proteins wereexpressed in almost all living beings, and not only in response to heat, but also when the cellwas exposed to a series of other stressing factors (toxic concentrations of metals, organicpollutants, temperature, osmolarity, hypoxia/anoxia and ultraviolet radiation), and thenthey began to be called stress proteins or anti-stress proteins (Meyer & Silva, 1999).Many toxicants affect the correct conformation and consequently, the function of differentproteins. In this condition, where the proteins are found incorrectly folded inside the cell, it

is initiated a stress response. The HSPs take action, acting as molecular chaperones, sincethey bind to other proteins, regulating their conformation, movement through themembrane or organelles and enzymatic activity (Calabrese et al., 2005). Therefore, theyavoid incorrect interactions between proteins, helping in their synthesis, folding anddegradation (Meyer & Silva, 1999).According to Bierkens (2000), HSPs are one of the main cellular markers in the evaluation ofthe toxicity of different compounds and are widely used to monitor ecosystems. Suchmonitoring has shown high levels of HSPs in the tissues of invertebrates collected incontaminated areas, when compared to those animals existent in uncontaminatedenvironments (Bierkens, 2000; Malaspina & Silva-Zacarin, 2006).These proteins are found highly conserved in all the living organisms (Burdon, 1986) andcan be classified according their molecular weight into four families: HSP90 (90 kDa), HSP70(70 kDa), HSP60 (60 kDa) and small HSPs. The family HSP70 is one of the most studied andseveral studies have shown its induction in stress conditions by heavy metals (Khler et al.,1992, 1996b; Nadeau et al., 2001; Zanger et al., 1996).Monari et al. (2011) worked with the mollusc species Chamelea gallina, observing an increasein the expression of HSP70. The authors affirm that the induction of HSP70 can beconsidered an adaptation mechanism associated with changes in the environmentalparameters.Silva-Zacarin et al. (2006), using immune-histochemical methods, observed an increase inthe levels of the products of the positive reaction to HSP70 in the salivary glands of bees

treated with acaricides in comparison with the control group. Moreover, they also verifiedalterations in the immune-reactivity between the nucleus and cytoplasm according to theacaricide used and the treatment period. According to the authors, the determination andlocation of HSP70 by immune-histochemistry can be useful to detect cellular responses tochemical stressors.Khler et al. (1992) points out the HSP70 in invertebrates of soil as a possible tool in themonitoring of environmental toxicants. In a study conducted by Zanger et al. (1996), theauthors exposed adult of the diplopod Julus scandinavius to substrates contaminated withdifferent concentrations of cadmium and investigated the expression levels of HSP70. Theanalyses showed that an increase in the concentration of cadmium in the animals dietresulted in high levels of HSP70.


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According to Zanger et al. (1996), the so-called hepatic cells of diplopods can present astrong expression of HSP70 in stress conditions induced by heavy metals. These cells arefound disperse through the cells of the fat body layer that compose the midgut and one oftheir functions is to help in the detoxification of the organism (Hopkin & Read, 1992).

In a study carried out by Nadeau et al. (2001), it was investigated the feasibility of usingHSP70 as marker of the presence of soil toxicants in the midgut of the earthworm L.terrestris. The authors concluded that HSP70 can be efficiently used in the assessment of thetoxicity of soils using test organisms exposed under laboratory conditions.

5. Conclus ion

Due to the constant release of harmful substances into the terrestrial environment, it isnecessary to know their action on the organisms present there, in order to avoid triggering apossible unbalance in the ecosystems. In this sense, we tried to present potentialbioindicators and biomarkers for ecotoxicological analyses of the soil. It is important to

highlight that the choice of the bioindicator organism is essential to the success ofenvironmental monitoring. Moreover, the combined use of morphological, biochemical andgenotoxic methods in the evaluation of injuries in sentinel species is interesting since itprovides a more complete understanding of the action of contaminants on organismsexposed, besides providing a greater reliability to the results obtained in the researches. Webelieve that understanding the importance of the combined use of different methodologiesin assessing the toxicity of substrates will be highly beneficial for the future work ofresearchers in Ecotoxicology.

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